1.1 Field of Invention
The present invention relates generally to antimicrobial agents, more particularly to antimicrobial peptides, and most specifically to branched cationic antimicrobial peptides.
1.2 General Background
Increasing bacterial resistance to conventional antibiotics has spurred research for novel antimicrobial agents. One such area concerns antimicrobial peptides (Hancock, 1999; Hancock and Lehrer, 1998, Lehrer and Ganz, 1996). Cationic peptides have an important role in defending the host against invading microbial organisms in both plants and animals (Otvos, 2000; Otvos et al., 2000). The activity of some cationic peptides are restricted to either gram positive or gram negative bacteria while others are active against both. Cationic peptides are also effective against fungal and viral infection Hancock, 1999).
Cationic peptides are small, 12-35 amino acids, diverse both in sequence and structure, and often possess a net positive charge due to the presence of arginine and lysine (Hancock, 1999). Since living organisms lack the ability to synthesize branched peptides all cationic microbial peptides are linear and this limits antimicrobial activity. Four major classes of antimicrobial peptides by structure are recognized: β-stranded, e.g. defensins and protegrins; α-helical, e.g. magainins and cecropins; extended coil, e.g. indolicidin and bac 5; and loops, e.g. bacteninin and polymyxins (Hancock, 1997).
1.3 Discussion of Prior Art
Patent literature has described microbial activity by several cationic peptides: WO 8900199; WO 885826, WO 8604356, EP 193351, EP 85250, U.S. Pat. Nos. 6,465,429, & 5,912,230. Helicity, hydrophobicity, and charge are considered important to cationic peptide selectivity toward prokaryotic membranes (Dathe and Wieprecht, 1999; Hoover et al., 2001; Hughes, 1999).
In addition to cationic amino acids cysteine, proline, glycine, histidine, and hydrophobic amino acids appear to have a structural functional role in selected microbial peptides (Ibid., Epand and Vogel, 1999; La Rocca et al., 1999; Oppenheim et al., 1998a; Sitaram and Nagaraj, 1999). Histidine, for example, is not present in many antimicrobial peptides but is found in the saliva in one group of low molecular weight linear peptides: Histatin 1 and 3 (Oppenheim et al., 1998b; Sabatini and Azen, 1989; Tsai and Bobek, 1998). Proteolytic fragments of Histatin 1 and 3 have been shown to have antifungal activity (Ibid.).
Defensins and protegrins are the primary antimicrobial peptides in humans wherein the former is abundant in phagocytes and small intestinal mucosa. Increases in serum defensins in non-neutropenic patients with sepsis have been observed and it has been suggested that defensins play a role in host defense against severe sepsis (Thomas et al., 2002). The role antimicrobial peptides have in host protection against systemic infections is not clear but the role played in prevention and control of local infections, particularly in higher organisms, is well evidenced.
Prokaryotes have several properties heightening sensitivity to cationic microbial peptides in comparison with eukaryotes. The almost universal negative charge on cell membranes and walls of bacteria is considered responsible for the antibacterial activity of cationic peptides. This charge is partly due to certain components: lipopolysaccharide (LPS) and anionic lipids in membranes; peptidoglycans and techoic acid in walls. Eukaryotic cell walls, in contrast, lack anionic lipids. A lack of cholesterol and lesser potential for membrane transfer also increase the sensitivity of prokaryote bacteria in comparison with eukaryotes and the combination of these properties enable cationic peptides to target and klll the former. At high concentrations, however, cationic peptides may be toxic to eukaryotic cells.
Gram negative bacteria have a peptidoglycan layer between the inner and outer cell membranes. Cationic peptides first bind to the negatively charged LPS in the outer membrane and then bind to the anionic lipids of the inner membrane in a self promoting mechanism. Both actions require a positive charge and this also enables penetration of the single inner membrane of gram positive bacteria. A sufficient number of cationic amino acids with a positive charge at physiologic pH is clearly necessary. Two mechanisms have been suggested for this: formation of a trans-membrane pore; and membrane solubilization. The latter appears to be the primary mechanism for inhibiting prokaryotic bacterial growth (Bechinger et al., 1999; Oren et al., 1999; Oren eta al., 2002; Oren and Shai, 1998) while the provision of a cationic cleavage peptide by lactoferrin (Vogel et al., 2002) supports the former. It has been suggested, moreover,that these mechanisms are not mutually exclusive and that not all antimicrobial peptides act by the same mechanism (Ibid.) In addition to these prokaryotic membrane destruction mechanisms β-defensins may enhance host defenses by reducing endotoxin levels (Giacometti et al., 2001) or by interacting with chemokine receptors (Yang et al., 2002).
Some bacteria evidently have outer membranes that are impenetrable by cationic peptides (Hancock, 1997; Preschel and Collins, 2001) and while synergism between several different type of antibiotics and cationic peptides has been shown in pre-clinical models (Hancock, 1999) and IB-367 has been evaluated in phase II clinical trials on oral mucositis (Bellm et al., 2000; Mosca et al., 2000) little is known about acute or chronic toxicity of may cationic peptides administered intravenously.
Defensins are similar to neurotoxic venoms in being small cationic peptides with cysteine bridges (Bontems et al., 1991; Kourie and Shorthouse, 2000) but are dissimilar in not affecting ion channel activity. Many defensins (Harder et al., 2001; Jia et al., 1999; Mallow et al., 1996) and neurotoxins (Blanc et al., 1996; Cruz et al., 1987; Gilles et al., 2000; Martin and Rochat, 1984) have histidines in close proximity with cationic amino acids and since the role of histidines in neurotoxins has not been investigated this similarity urges caution in development of cationic peptides de novo.
Non-viral gene therapy carriers are also cationic, often contain arginine and lysine, and the resulting positive charge is important to both interaction with DNA and the cell surface. Branched polymers have been made primarily from histidine and lysines (Chen et al., 2001, WO 147496) that, in contrast to cationic liposomes, are effective carriers of DNA into cells. The role of histidine in these non-viral gene therapy carriers is thought to be the buffering of endosomes but endosomes have no role in the antimicrobial activity of cationic peptides.
1.4 Statement of Need
The similarity of defensins to neurotoxins in being linear small cationic peptides with cysteine bridges has been noted as urging caution in the development of cationic peptides de novo for use as an antibiotic. Toxicity to cardiac and neural cells is recognized as a grave concern particularly for a systemic antibiotic for both natural and de novo peptides.
While the prior art has, as related above, demonstrated widespread success of defensins possessing cationic amino acids in fighting local infections in vivo and of cationic peptides as an antimicrobial in vitro, the development of an effective antimicrobial peptide with minimal toxcity is essential.
The demonstrated ability of bacteria in recent years to develop resistance to all known antibiotics, including, most recently and most alarmingly, strains resistant to vancomycin, the antibiotic of last resort, is a problem of vast dimension. Bacterial infection has, again, become a primary concern of surgical medicine. And while for obvious reasons the phenomenon has not been advertised, hospitals have become reservoirs for resistant bacteria. A poignant need is therefore recognized for an alternative to conventional systemic antibiotics that will not incur the development of bacterial resistance.